Molecular Phenotype of the np 7472 Deafness-Associated Mitochondrial Mutation in Osteosarcoma Cell Cybrids

Abstract

The nucleotide pair (np) 7472 insC mitochondrial DNA mutation in the tRNA(Ser)(UCN) gene is associated with sensorineural deafness, combined in some individuals with a wider syndrome including ataxia and myo-clonus. Previous studies in osteosarcoma cell cybrids revealed only a mild respiratory defect linked to the mutation. We have investigated the biochemical and molecular consequences of the mutation, using a panel of seven osteosarcoma cell cybrids containing 100% mutant mtDNA, plus two cybrids carrying 100% wild-type mtDNA from the same patient. The mutation is associated with a mild growth deficit in selective (galactose) medium that is only significant in combination with a reduced mtDNA copy number, suggesting a mechanism that might modulate clinical phenotype. The mutation results in a 65% drop in the steady-state level of tRNA(Ser)(UCN), but causes at most only a very mild and quantitative abnormality of mitochondrial protein synthesis, associated with modest hypersensitivity to doxycyclin. No evidence for a specific defect in aminoacylation was obtained, and unlike the case with the np 7445 mutation, the pattern of RNA processing of light strand transcripts of the ND6 region was not systematically altered. Comparing the np 7472 and np 7445 mutant phenotypes in cultured cells suggests that sensorineural deafness can result from a functional insufficiency of mitochondrial tRNA(Ser)(UCN), to which some cells of the auditory system are especially vulnerable.

The 7472insC Mitochondrial DNA Mutation Impairs the Synthesis and Extent of Aminoacylation of tRNASer(UCN) but Not Its Structure or Rate of Turnover

“…Effects on mitochondrial protein synthesis are minimal, with only a slight quantitative decrease in mitochondrial translation products detectable by pulse labeling (19), an effect mildly exacerbated by doxycycline treatment (19). The only clear molecular effect of the mutation that can be seen in cybrid cells is a decrease of ϳ70% in the steady-state level of tRNA Ser(UCN) (19), an effect shared with the A7445G deafness-associated mutation (20,21). Unlike the case of the latter, which has been studied only in lymphoblastoid cells, 7472insC produces no systematic change to the level of the upstream (ND6) mRNA, leading to the suggestion that its effects are most likely on the half-life of tRNA Ser(UCN) rather than on its processing (19).…”

“…Clinically, most subjects have an apparently non-syndromic hearing impairment (16), with a minority suffering a more widespread neurological disease including ataxia and myoclonic seizures (16 -18), sometimes with a measurable deficit of cytochrome c oxidase (18). In 143B osteosarcoma-derived cybrid cells, homoplasmy for the 7472insC mutation produces only a very modest biochemical phenotype, comprising a small decrease in complex I activity (17) and a growth deficit in galactose medium when the mutation is present in combination with a diminished copy number of mtDNA (19). Effects on mitochondrial protein synthesis are minimal, with only a slight quantitative decrease in mitochondrial translation products detectable by pulse labeling (19), an effect mildly exacerbated by doxycycline treatment (19).…”

“…In 143B osteosarcoma-derived cybrid cells, homoplasmy for the 7472insC mutation produces only a very modest biochemical phenotype, comprising a small decrease in complex I activity (17) and a growth deficit in galactose medium when the mutation is present in combination with a diminished copy number of mtDNA (19). Effects on mitochondrial protein synthesis are minimal, with only a slight quantitative decrease in mitochondrial translation products detectable by pulse labeling (19), an effect mildly exacerbated by doxycycline treatment (19). The only clear molecular effect of the mutation that can be seen in cybrid cells is a decrease of ϳ70% in the steady-state level of tRNA Ser(UCN) (19), an effect shared with the A7445G deafness-associated mutation (20,21).…”

Multiple origins of the mtDNA 7472insC mutation associated with hearing loss and neurological dysfunction

“…It has been shown that the mutation causes a 65% reduction in levels of tRNA Ser(UCN) although it results in only a small decrease in mitochondrial respiratory enzyme activities. 3,8 Why this defect appears most pronounced in the inner ear is not yet known.…”

Mitochondrial rRNA and tRNA and hearing function

“…Biochemical data showed that cell lines carrying this mutation produced a significant decrease in the level of this tRNA, which was combined with a small but clear decrease in the extent of aminoacylation. Only very subtle effects on structure or stability were found [89,90].…”